Understanding what regulates variations in synaptic strength is of great interest, since changes in synaptic function are a hallmark of many psychiatric and neurological diseases. At glutamatergic synapses, AMPA and NMDA receptors (NMDARs) are positioned across from presynaptic active zones by retention within the postsynaptic density (PSD). It is well established that altering the number of postsynaptic receptors is a key mechanism of synaptic plasticity. However, theory and computational modeling suggest that aside from the importance of receptor number, the distribution of receptors within the PSD may have a dramatic impact on synapse physiology, by governing the likelihood that released neurotransmitter will activate the receptors. In my lab, single-molecule mapping of the PSD (using an imaging technique called PALM) has led to the identification of nanodomains within the PSD that contain a relatively high density of the scaffold protein PSD95 and the NMDAR subunit GluN2B. These data suggest that the nanoscale organization of the synapse could play a critical role in synaptic function. Thus, I hypothesize that nanodomains of PSD-95 control receptor activation through subsynaptic localization of GluN2B. Previously, it has not been feasible to test this idea in live cells due to the lack of imaging resolution. However, by combining our recently published assays with a new approach I have developed, I propose to test this how this novel feature influences synaptic transmission. To determine the relationship between PSD nanoscale organization and NMDAR activation, I co-transfected cultured hippocampal neurons with a photoactivatable version of PSD-95 and the genetically encoded Ca2+ indicator GCaMP6f. This permitted me to combine PALM imaging of synapse nanostructure with measurement of miniature spontaneous Ca2+ transients in the same individual spines. This approach revealed a significant correlation between NMDAR- mediated Ca2+ transients at single spines and the fractional area of the PSD that was within nanodomains. There was no relationship between the Ca2+ transient and PSD area, consistent with previous observations that synaptic NMDAR number is only weakly correlated with PSD size. To better understand the mechanism responsible for this relationship, I will first use a combination of imaging and electrophysiological measures of NMDAR function at synapses whose nanostructure is monitored via PALM. Second, GluN2A and GluN2B receptor subtypes are predicted to vary in the spatiotemporal aspects of their activation, and GluN2B is preferentially enriched in PSD-95 high-density nanodomains. Therefore, I will probe the relative contribution of each subunit to the relationship between receptor activation and PSD nanostructure. Finally, I will assess the role of NMDAR interaction with PSD-95 using peptides or PSD-95 mutants that cannot bind NMDARs, each of which I expect to abolish or weaken the relationship between nanostructure and NMDAR activation. Fine tuning of NMDAR activation through nanoscale manipulations of receptor distribution could have a profound impact on synaptic physiology and the induction of synaptic plasticity.